BIOSYNTHESIS OF THE SIDE-CHAIN OF YEAST GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORS IS OPERATED BY NOVEL MANNOSYLTRANSFERASES LOCATED IN THE ENDOPLASMIC-RETICULUM AND THE GOLGI-APPARATUS

Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain three different types of side chains attached to the alpha 1,2-linked mannose of the conserved prot ein-ethanolamine-PO4-Man alpha 1,2Man alpha 1,6Man alpha 1,4Glc glycan core. The possible side chains are Man alpha 1,2- or Man alpha 1,2Man alpha 1,2- or Man alpha 1,3Man alpha 1,2- (Fankhauser, C., Homan, S. W., Thomas Gates, J. E., McConville, M. J., Desponds, C., Conzelmann, A, and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365-26374). To det ermine in what subcellular compartment these side chains are made, we metabolically labeled GPI anchored membrane proteins with myo-[2-H-3]i nositol in secretion mutants blocked at various stages of the secretor y pathway and analyzed the anchor structure of the labeled glycoprotei ns. When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors c ontained a side chain consisting of a single alpha 1,2-linked mannose. GPI proteins trapped in the cis-Golgi of sec7 contained Man alpha 1,3 Man alpha 1,2- but no Man alpha 1,2Man alpha 1,2- side chains. Mutants blocked at later stages of the secretory pathway made increased amoun ts of side chains containing two mannoses. Man alpha 1,2Man alpha 1,2- and Man alpha 1,2Man alpha 1,2- side chains were preferentially assoc iated with ceramide- and diacylglycerol containing GPI anchors, respec tively. Mnn1, mnn2, mnn3, mnn5, and mnt1(= kre2), i.e. mutants which lack or down-regulate 1,2- and 1,3-mannosyltransferases used in the el ongation of N . and O-glycans in the Golgi, add the fifth mannose to G PI anchors normally. The same conclusion was reached through the analy sis of deletion mutants in KTR1, KTR2, KTR3, KTR4, and YUR1 which all are open reading frames with high homology to MNT1. Mutants deficient in the Golgi elongation of N-glycans such as anp1, van1, mnn9 are defi cient in the maturation of the N-glycans of GPI-anchored glycoproteins , but process the GPI anchor side chain normally. Data are consistent with the idea that the fourth mannose is added to proteins as part of the anchor precursor glycolipid in the endoplasmic reticulum, whereas the fifth mannose is added by not yet identified alpha 1,3- and alpha 1,2-mannosyltransferases located in the Golgi apparatus.